Billions of dollars are spent each year in efforts to control insects that damage crops and threaten food supplies. One alternative to the use of synthetic chemical pesticides are naturally occurring insecticidal crystal protein (Cry) toxins from the bacterium Bacillus thuringiensis (B.t.). In order to preserve Cry proteins as a viable option for pest control in years to come, efforts are being made to prevent their over-use, as the development of resistance to Cry proteins by some insect strains has been observed under certain conditions. The two main insects that are currently known to develop resistance to Cry proteins are the diamondback moth (DBM; Plutella xylostella) and the tobacco budworm (Heliothis virescens).
In understanding how these and other insects might develop resistance to Cry proteins, the mechanism(s) of action of Cry proteins is being investigated. Specific binding to insect midgut receptors is a key step in the mode of action of Cry proteins. Despite exceptions [1], in most cases Cry toxin specificity and potency correlate with the extent of toxin binding to midgut brush border membrane receptors in vitro [2, 3]. Effective toxin binding to receptors results in toxin insertion and oligomerization on the midgut cell membrane, leading to pore formation and cell death by osmotic shock [4].
In brush border membrane vesicles (BBMV) from Heliothis virescens larvae, three groups of binding sites (A, B, and C) for Cry1A toxins were proposed based on their toxin binding specificities [5, 6]. The A binding sites, which bind Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa and Cry1Ja toxins, include the cadherin-like protein HevCaLP (Jurat-Fuentes et al., in preparation) and a 170-kDa N-aminopeptidase (APN) [5, 7-9]. Currently, there is evidence that both HevCaLP [10] and the 170-kDa APN [8, 10] function as Cry1A toxin receptors, and knockout of HevCaLP, a protein predicted to function in cell adhesion processes, results in Cry1 resistance in larvae of YHD2 strains of H. virescens [10]. In the B binding site group, a 130-kDa protein has been shown to recognize both Cry1Ab and Cry1Ac. The C binding site group includes Cry1Ac toxin-binding proteins smaller than 100-kDa in size [5].
Cry1 toxin-binding proteins of 60- to 80-kDa in size have been described in toxin overlays of BBMV proteins from H. virescens [5], Manduca sexta [1], and Plodia interpunctella [12]. In 2D proteomic analysis of M. sexta BBMV proteins, McNall and Adang [13] reported Cry1Ac binding to a form of alkaline phosphatase (ALP, EC 3.1.3.1). Membrane bound ALP from Bombyx mori and M. sexta are attached to the brush border cell membrane by a glycosylphosphatidylinositol (GPI) anchor [13-15]. Specific interactions between Cry1Ac and ALPs under native conditions resulting have been reported for M. sexta [16] and H. virescens [17].
Altered glycosylation of 63- and 68-kDa glycoproteins was proposed as the reason for reduced binding of soybean agglutinin (SBA) in H. virescens YHD2 strain, which are resistant to Cry1Ac [11]. However, a correlation between a reduction in the amount of the 68 kDa protein and the development, by insects, of resistance to B.t. Cry proteins was never before suggested or investigated. Furthermore, a link between membrane-bound alkaline phosphatases (and associated levels of enzyme activity) and the development of resistance by insects to Cry proteins has never been proposed or suggested.